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Axl-specific antibodies and uses thereof

A specificity, antibody technology, applied in the direction of antibodies, antibody mimics/scaffolds, preparations for in vivo tests, etc., can solve problems such as lack of sensitivity

Pending Publication Date: 2020-06-09
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] A disadvantage of therapeutic antibodies that reduce AXL activity is their potential to encounter therapeutic resistance, the nature of which is either innate or acquired, where innate resistance is due to an inherent lack of sensitivity , acquired resistance is due to bypass mechanisms that arise during selection pressure for treatment

Method used

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  • Axl-specific antibodies and uses thereof
  • Axl-specific antibodies and uses thereof
  • Axl-specific antibodies and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0137] Embodiment 1: Preparation of anti-AXL antibody

[0138] Monoclonal antibodies (mAbs) against AXL were generated by immunizing mice with the extracellular domain of recombinant human AXL protein (rhAXL-ECD; SEQ ID NO:58) and by genetic immunization.

[0139] Preparation of rhAXL-ECD: The pTT5 construct containing a synthetic recombinant fragment of the C-terminal His8-tagged recombinant human (rh)AXL extracellular domain (ECD; SEQ ID NO:71) was expressed in CHO cells by Ni-agarose Purified and verified by SDS-PAGE (data not shown).

[0140] Immunization: Mouse mAbs were generated by genetic immunization. For genetic immunization, mice were bled (pre-immune serum) and immunized on days 0 and 42 with 100 μg of pTT40 expression plasmid for expression of soluble rhAXL-ECD by hydrodynamic tail vein delivery (HTV). After 7-10 days, mice were bled, and serum titers were measured by ELISA. After 4-5 months, a booster injection with 100 μg pTT40-AXL-H8G was done by HTV 3-4 d...

Embodiment 2

[0144] Example 2: Evaluation of cross-reactivity of anti-AXL mAbs

[0145] The anti-hAXL monoclonal antibodies purified in Example 1 were evaluated for their cross-reactive binding properties to the recombinant AXL extracellular domain.

[0146] To evaluate the cross-reactivity of mAbs with other members of the TAM receptor tyrosine kinase subfamily, the binding of the mAbs to rhAXL-ECD, rhMER-ECD, rhTyro-3 and BSA (negative control) was measured by ELISA. 96-well half-area plates (Costar) were coated with 25 μl of 5 μg / ml rhAXL-ECD, rhMER-ECD, rhTyro-3 or BSA in PBS and incubated overnight at 4°C. Microplates were washed 3 times with PBS, blocked with PBS-BSA 1%, added 25 μl of 10 μg / ml diluted in PBS-BSA 1%, and incubated at 37 °C, 5% CO 2 Incubate for 2 hours. with PBS-TWEEN TM 20 Wash plates 4 times at 0.05% at 37°C, 5% CO 2 Incubate for 1 hour with 25 μl of secondary antibody alkaline phosphatase-conjugated F(ab)'2 goat anti-mouse IgG (H+L) (Jackson Immunoresearch)...

Embodiment 3

[0150] Example 3: Functional characterization of ADC potential

[0151] The anti-hAXL-ECD monoclonal antibody purified in Example 1 was evaluated for its internalization ability and antibody-drug conjugate (ADC) potential in an ADC surrogate screening assay. Non-small cell lung cancer (NSCLC) A549 cells, breast cancer MDA-MB231 cells or glioblastoma U87MG cells, all expressing moderate to high levels of AXL, were used. Selection showed low or sub nM (IC 50 ) efficacy of mAbs.

[0152] cell culture. The following cell lines were obtained from ATCC and cultured according to the supplier's recommendations: A549 human non-small cell lung cancer (NSCLC), MDA-MB-231 triple-negative (ER- / PR- / HER2 low) breast cancer (TNBC) and U87MG Glioblastoma cell line. Typically, cells were passaged once or twice a week and used within 4-6 weeks for all experiments.

[0153] Evaluation of antibody-mediated cytotoxicity as antibody-drug conjugates. Primary mouse antibody (typically 1 nM con...

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Abstract

AXL-specific antibodies and uses therefor are described, including monoclonal and single domain antibodies. Such antibodies bind to cell surface expressed human AXL at an epitope in an immunoglobulin-like (IgL) domain of the AXL ectodomain. The antibody may be used in an antibody-drug conjugate (ADC), for example in the treatment, detection or staging of cancer. The antibody may be biparatopic.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Patent Application 62 / 557,870, filed September 13, 2017, which is incorporated herein by reference. technical field [0003] The present invention relates to AXL-specific antibodies and uses thereof. Such antibodies bind cell surface expressed human AXL and can be used in fusions or in antibody-drug conjugates. Background technique [0004] AXL is a member of the Tyro3-AXL-Mer (TAM) receptor tyrosine kinase subfamily. A schematic diagram of the AXL domain structure is in figure 1 shown in the prior art illustration. The extracellular domain of the AXL protein (100) was shown with the indicated exons (1-20). The AXL protein (100) comprises 2 immunoglobulin-like domains (102) and 2 fibronectin type III (FNIII) repeat regions (104). The transmembrane domain (106) spans the cell membrane, followed by the intracellular kinase domain (108). Splicing (i...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61K47/68A61K49/00A61P35/00C07K14/705C07K19/00C12N15/113C12N15/13C12N5/10C12N7/01C12P21/08G01N33/53G01N33/543
CPCG01N33/543A61K47/6803A61K47/6849A61P35/00G01N33/573G01N2333/705G01N2333/9121C07K16/2863C07K16/30A61K2039/505C07K2317/22C07K2317/24C07K2317/31C07K2317/33C07K2317/569C07K2317/73C07K2317/77C07K2317/92A61K47/6851C07K14/71C07K2319/00C07K2319/41C07K2319/42A61K47/68031A61K47/68033A61K49/00C07K2317/565C07K2317/567C12N5/16
Inventor K·亨利M·L·贾拉米洛C·R·麦肯齐A·马西尔
Owner NAT RES COUNCIL OF CANADA
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